P-036: PIEZO1 ACTIVATION AUGMENTS SICKLING PROPENSITY AND THE ADHESIVE PROPERTIES OF SICKLE RED BLOOD CELLS
Authors: CONRAN N., EGÉE S., HATERN A., LEONARDO F., BRITO P., COSTA F., BOISSON C., JOLY P., RENOUX C., POUTREL S., GAUTHIER A., NADER E., CONNES P.
Journal: HemaSphere (2022)

Purpose: Sickle cell disease (SCD) alters the hemoglobin molecule, modifying the properties of red blood cells (RBC) and causing their destruction in the circulation, leading to multiple complications. The increased adhesive properties and reduced deformability of sickle RBC make a major contribution to the vaso-occlusive processes that characterize the disease. Piezo1 is a mechanosensitive membrane protein that is activated by changes in vascular flow, triggering calcium (Ca2+) influx into cells. We aimed to identify a role for Piezo1 in altering the adhesive properties of sickle RBC and their propensity for sickling, and the contribution of Ca2+ influx and Gardos Channel (GC) activation to these alterations. Materials and methods: Peripheral blood was collected, with informed consent, from patients with HbSS SCD, cared for at the Hospices Civils de Lyon and Institut d’Hématologie et d’Oncologie Pédiatrique de Lyon, France, and at the Hematology Center, University of Campinas, Brazil. Piezo1 activation on separated RBCs was achieved by incubation of cells with Yoda1 (0.1-10µM), a Piezo1 agonist. RBC membrane protein expression and Ca2+ influx were observed by flow cytometry, microfluidic adhesion assays were used for examining the adhesive properties of cells and RBC deformability was monitored using ektacytometry under oxygen gradient. Channel activity under Piezo1 activation was monitored using change in membrane potential method (CCCP). Results: Oxygen gradient ektacytometry demonstrated that activation of Piezo1 on sickle RBC reduces cellular deformability and increases the sickling propensity of sickle RBC during deoxygenation. In contrast, inhibition of Piezo1 with GsMTX4 increased the deformability of sickle RBC and decreased their sickling propensity. Adhesion assays indicated that activation of Piezo1 was associated with a significant increase in the ability of sickle RBC to adhere to laminin under flow conditions. While flow cytometry demonstrated a clear effect of Piezo1 activation by Yoda1 on phosphatidylserine exposure on sickle RBC, accompanied by Ca2+ influx, no alteration in the expression of the laminin-binding BCAM adhesion molecule was observed on these cells. However, co-incubation of cells with a BCAM function-neutralizing antibody molecule abolished the Yoda1-dependent increase in sickle RBC adhesion, indicating a role for BCAM adhesion molecule activation (rather than expression) in this mechanism. Importantly, membrane potential measurement showed that Piezo1 stimulation triggers pronounced membrane hyperpolarization linked to GC activation, a phenomenon exacerbated by low oxygen partial pressure; furthermore, the increase in RBC sickling propensity following Piezo1 activation occurred in a GC-dependent manner (as demonstrated by co-incubation with TRAM34, a GC inhibitor). Conclusion: In conclusion, pharmacological activation of Piezo1 on the sickle RBC surface significantly modulates the function and properties of these cells, suggesting that this mechanosensor may represent a potential therapeutic target molecule in SCD. The authors do not declare any conflict of interest